The application of comparative proteomic analysis to screen proteins associated with mechanical properties of engineered cartilage: a preliminary study / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study proteins correlated with the mechanical properties of engineered cartilage by screening significantly changed proteins during cartilage formation by comparative proteomic analysis.</p><p><b>METHODS</b>Human chondrocyte, cultured and expanded, were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffolds. After 4 weeks of culture in vitro, the constructs were divided into three groups. There were 6 specimens in each group. For the regular in vitro culture group (A), the constructs were kept in culture at the original condition for an additional 6 weeks. For in vivo groups, the constructs were implanted subcutaneously into nude mice for either 6 weeks (B) or 12 weeks (C). All specimens were harvested for gross observation, average wet weight and volume measurement, histology, immunohistochemistry and biomechanics to evaluate the results. Meanwhile, comparative proteomic analysis was performed for each group, and those proteins involved in extracellular matrix with at least 2 folds up-regulation were chosen for further exploration. The correlations between Young's modulus and the relative content of the selected proteins were analyzed by Pearson correlation coefficient.</p><p><b>RESULTS</b>All these samples in the three groups eventually formed hyaline-like cartilage structure. Specimens in C and B groups were similar with adult articular cartilage in appearance, and had multiple mature lacuna in histology. However, those specimens in A group had loose texture with irregular hypertrophy lacuna. Specimens implanted for 12 weeks in vivo had better wet weight (372.5 +/- 35.4) mg and Young's modulus (8.68 +/- 2.65) MPa than those cultured in vivo for 6 weeks (346 +/- 34.5) mg, (3.25 +/- 1.24) MPa (P < 0.01). In group A, they were (184.4 +/- 12.28) mg and (0.7 +/- 0.23) MPa. This study had detected 44 proteins in ECM by comparative proteomic analysis, then chosing the greatest ratio of 6 up-regulation proteins compared between C and A groups. The correlation results indicated the content of Decorin, Chondroadherin and Fibromodulin were linear correlation with the mechanical properties of engineered cartilage (P < 0.05).</p><p><b>CONCLUSIONS</b>Comparative proteomic analysis could provide large scale information of associated proteins, making it profit for advanced research on the relationship between extracellular matrix and mechanical properties of engineered cartilage by combination with tissue reconstruction techniques.</p>

Preliminary study of in vitro chondrogenesis by co-culture of chondrocytes and adipose-derived stromal cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of in vitro chondrogenesis by co-culture of chondrocytes and adipose-derived stromal cells (ADSCs) so as to confirm the hypothesis that chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs.</p><p><b>METHODS</b>Human ADSCs and porcine auricular chondrocytes were in vitro expanded respectively and then were mixed at the ratio of 7:3 (ADSCs: chondrocytes). 200 microl mixed cells (5.0 x 10(7)/ml) were seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold, 8 mm in diameter and 2 mm in thickness, as co-culture group. Chondrocytes and ADSCs with the same cell number were seeded respectively onto the scaffold as positive control group and negative control group. 200 microl chondrocytes (1.5 x 10(7)/ml) were seeded as low concentration chondrocyte group. There were 6 specimens in each group. All specimens were harvested after in vitro culture for 8 weeks in DMEM plus 10% FBS. Gross observation, histology, immunohistochemistry, wet weight measurement and glycosaminoglycan (GAG) quantification were used to evaluate the results. Multiple-sample t-test statistics analysis was done to compare the difference of wet weight and glycosaminoglycan(GAG) content between the groups.</p><p><b>RESULTS</b>Cells in all groups had fine adhesion to the scaffold and could secrete extracellular matrix. In co-culture group and positive control group, cell-scaffold constructs could maintain the original size and shape during in vitro culture. At 8 weeks, cartilage-like tissue formed in gross appearance and histological features, and abundant type II collagen could be detected by immunohistochemistry. Wet weight and glycosaminoglycan(GAG) content of co-culture group were respectively (174 +/- 12) mg and (7.6 +/- 0.4) mg. There were respectively 75% (P < 0.01) and 79% (P<0.01) of those of positive control group. In negative control group, however, constructs shrunk gradually without mature cartilage lacuna in histology. In low concentration chondrocyte group, constructs also shrunk obviously with small amount of cartilage formation at the edge area of the construct, and wet weight was (85 +/- 5) mg, which was 37% (P<0.01) of that of positive control group.</p><p><b>CONCLUSIONS</b>Chondrocytes can provide chondrogenic microenvironment to induce chondrogenic differentiation of ADSCs and thus promote the in vitro chondrogenesis of ADSCs.</p>

Influence of in vivo or vitro microenvironment on the mechanical properties and histological structure of tissue engineered cartilage / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the influence of in vivo or vitro microenvironment on the mechanical properties and histological structure of tissue engineered cartilage, and to provide the appropriate parameters for cartilage construct in vitro.</p><p><b>METHODS</b>Human fetal articular chondrocytes were cultured and expanded in vitro, the passage 2 chondrocytes were seeded at the density of 6 x 10(7) cells/cm3 to cylindric dimensional scaffolds made by polyglycolic acid (PGA) and polylactic acid (PLA). These constructs were cultured in vitro for 4 weeks. After 4 weeks, part of samples were implanted subcutaneously into nude mice for 6 and 12 weeks, the others continued to be cultured in vitro. All specimens were harvested after 6 and 12 weeks, and evaluated by gross observation, histology, histochemistry, ultrastructure and mechanical test.</p><p><b>RESULTS</b>All specimens in vivo and vitro eventually formed good shape hyaline cartilage. The constructs in vivo group was white color with smooth surface, and had better mechanical properties than those in vitro, by TEM we can observe the thick and striated collagen fibers in regularly arranged collagen fibril which was similar to adult articular cartilage. The constructs in vitro group was yellow color with coarse surface, the appearance and ultrastructure was similar to fetal articular cartilage. Specimens implanted for 12 weeks in vivo had better compressive modulus(38.28 +/- 3.95) MPa and collagen diameter (41.58 +/- 2.78) nm than those cultured in vitro at the same time (4.12 +/- 0.63) MPa, (15.83 +/- 1.70) nm (P < 0. 01).</p><p><b>CONCLUSIONS</b>The structure and function of human tissue engineered cartilage became mature gradually from vitro to vivo, thick and striated collagen fibrils net similar to adult articular cartilage can be formed in constructs of vivo group,increased collagen cross-linking might be the reason that their mechanical properties been greatly improved.</p>

In vitro proliferation of rat Leydig cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of in vitro proliferation of rat Leydig cells by modifying the cell culture system.</p><p><b>METHODS</b>Leydig cells were isolated from three-week-old rats by a procedure combining collagenase dispersion, stainless steel mesh infiltration and differential adhesion. The isolated cells were cultured in DMEM/F12 and modified media for stem cell proliferation, and the proliferation of the cultured cells was evaluated by cell counting and MTP test. The expression of 3beta-HSD in the cultured cells was detected by immunohistochemistry and flow cytometry, and testosterone productivity in the isolated Leydig cells with or without hCG stimulation was determined at 2 hours and 4 days after cell isolation.</p><p><b>RESULTS</b>The Leydig cells cultured in the modified media proliferated actively, with a doubling time of (2.26 +/- .31) days, as compared with (16.32 +/- 2.14) days for those cultured in the traditional media (P <0.05). The 3beta-HSD positive rate in the cultured cells was (554.3 +/- 7.1)% after 2 hours and (93.6 +/- 4.6)% after 4 days of culture. All the proliferated cells exhibited testosterone productivity, and their testosterone secretion was significantly upregulated by hCG stimulation (P <0.05).</p><p><b>CONCLUSION</b>Leydig cells isolated by differential adhesion proliferate actively in the modified culture media.</p>

The study on the role of the soluble factors secreted by engineered cartilage in inducing bone marrow stromal cells chondrogenesis / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the role of the soluble factors secreted by tissue engineered cartilage in promoting bone marrow stromal cells (BMSCs) chondrogenesis as an important aspect.</p><p><b>METHODS</b>Porcine BMSCs, chondrocytes and dermal fibroblasts were respectively in vitro expanded and then seeded onto the polyglycolic acid/polylactic acid (PGA/PLA) scaffold. After 3 days, they were indirectly co-cultured by transwell. BMSCs-scaffold constructs were co-cultured with chondrocytes-scaffold constructs as experiment group (Exp), while co-cultured with fibroblasts-scaffold constructs as control group. BMSCs with the same cell number were seeded onto the scaffolds as another control group. There were 3 specimens in each group. All specimens were harvested after in vitro indirect co-culture for 8 weeks. Gross observation, histology, immunohistochemistry and RT-PCR were used to evaluate the results.</p><p><b>RESULTS</b>The BMSCs-scaffold constructs co-cultured with chondrocytes-scaffold shrunk gradually during in vitro culture, but formed the mature lacuna structures and metachromatic matrices, collagen II expression could be observed by immunohistochemistry and RT-PCR examination. In the control group, the constructs shrunk greatly during in vitro culture and showed mainly fibrous tissue.</p><p><b>CONCLUSIONS</b>The soluble factors secreted by chondrocytes can solely induce chondrogenic differentiation of BMSCs and thus promote the in vitro chondrogenesis of BMSCs.</p>

Preliminary study on tissue-engineered cartilage with human dermal fibroblasts co-cultured with porcine chondrocytes in vitro / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of constructing tissue-engineered cartilage with human dermal fibroblasts (HDFs) in vitro.</p><p><b>METHODS</b>Porcine articular chondrocytes and HDFs were isolated and in vitro expanded respectively. Then they were mixed at the ratio of 1:1 (chondrocytes: fibroblasts) . The mixed cells were seeded onto polyglycolic acid (PGA) scaffold at the ultimate concentration of 5.0 x 10(7)/ml as co-culture group. Chondrocytes and HDFs at the same ultimate concentration were seeded respectively onto the scaffold as chondrocyte group ( positive control group) and fibroblast group ( negative control group). The specimens were collected after in vitro culture for 8 weeks. Gross observation, histology and immunohistochemistry were used to evaluate the results.</p><p><b>RESULTS</b>In chondrocyte group, the cell-scaffold constructs could maintain the original size and shape during in vitro culture. The new formed cartilage-like tissue had typical histological structure and extracellular matrix staining similar to normal cartilage. In co-culture group the constructs shrunk slightly at 8 weeks, cartilage-like tissue formed and GAG could be detected for strong expression by Safranin O staining. Furthermore, using the specific identification, a few HDFs derived cells were found to form lacuna structure at the peripheral area of cartilage-like tissue. In fibroblast group, the constructs deformed and shrunk gradually without mature cartilage lacuna in histology.</p><p><b>CONCLUSION</b>The 3D-co-culture system can effectively induce the differentiation of HDFs to chondrocytes. The tissue-engineered cartilage can be constructed in vitro with the 3D-co-culture system.</p>

Construction of urethra mucosa structure in vitro by tissue engineering / 中华男科学杂志

<p><b>OBJECTIVE</b>To construct a structure of urethra mucosa in vitro by tissue engineering.</p><p><b>METHODS</b>Primary porcine urothelial cells (UC) were obtained from the porcine bladder by enzymatic digestion and detected by immunofluorescence and RT-PCR. Bladder acellular matrix grafts (BAMG) were prepared, used as the scaffold and then evaluated by HE staining, Masson's trichrome staining, immunohistology and scanning electron microscopy. After in vitro culture and amplification, the UCs were seeded on the luminal surface the BAMGs.</p><p><b>RESULTS</b>After 1 week of in vitro culture, the UCs formed a multilayer structure on the luminal surface of the BAMGs along the basement membrane. The tissue-engineered urothelium and BAMG complex was well formed and pan cytokeratins were positively expressed in the UCs on the scaffold.</p><p><b>CONCLUSION</b>By tissue engineering, the urethra mucosa structure can be rapidly constructed in vitro, which can be applied to the repair of such urethral defect as hypospadias and urethral stricture.</p>

Constructing of smooth muscle layers of tissue engineered blood vessel in a bioreactor / 中华整形外科杂志

<p><b>OBJECTIVE</b>To investigate the feasibility of in vitro constructing tissue engineered blood vessels in bioreactor.</p><p><b>METHODS</b>Cell-PGA (polyglycolic acid) complex was constructed by seeding smooth muscle cells isolated from canine carotid artery on PGA unwoven fibers. The cell-PGA complex was cultured in a bioreactor (pulse rate = 75/min, radical distension < 5%). After three or six weeks in vitro culture, engineered tissues were harvested and tested.</p><p><b>RESULTS</b>Grossly,the experimental groups showed a tubular structure with a round lumen and good elasticity. Histological staining revealed smooth muscle fibers layers and dense elastic fibers presented in the engineered vessel wall. Bands of smooth muscle fibers and continuous endothelial cells layer were detected by the immuno-histological staining. In contrast, the control group took on poor elasticity, collapsed lumen and pale surface in the gross observation. In addition, its arrangement of smooth muscle fibers and elastic fibers was random and disorganized by histological observation, which was also confirmed by the immunohistological staining. The structure of 6 weeks TEBVs was more mature than that of 3 weeks and the biomechanical property of dynamic ones was as much as 60% of the normal one.</p><p><b>CONCLUSIONS</b>Blood vessel with good elasticity can be constructed in a bioreactor by tissue engineering approach.</p>

Preliminary study of constructing tissue-engineered cartilage with the endoskeletal scaffold of HDPE by bone marrow stromal cells / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of using a nonreactive, permanent endoskeletal scaffold to create the prothesis in special shape which is covered with tissue-engineered cartilage.</p><p><b>METHODS</b>Porcine BMSCs and articular chondrocytes were isolated and expanded respectively in vitro. Porcine BMSC of passage 1 in the concentration of 10 x 10(7)/ml were seeded onto a cylinder-shaped PGA (1 mm in thickness)/Medpor (3mm in diameter and 5mm in highness) scaffold as the experimental group. After the cell-scaffold constructs were cultured for 5 days, the primary medium, high-glucose DMEM medium with 10% fetal bovine serum (FBS), was replaced by chondrogenically inductive medium for 4 weeks. BMSCs and chondrocytes of the same concentration were seeded respectively onto the scaffold as the negative control group and the positive control group. After cultured in vitro for 4 weeks, the cell-scaffolds construct were implanted into subcutaneous pockets on the back of nude mice. Four and eight weeks later, the formed cartilage prosthesis were harvested and then evaluated by gross view, histology, immunohistochemistry and glycosamino-glycan (GAG) content.</p><p><b>RESULTS</b>Cells in all groups had fine adhesion to the scaffold and could secrete extracellular matrix. All specimens in experimental group and positive control group formed mature cartilage with collagen II expression.The mature catrtilage wraped HDPE compactly and grown into the gap of HDPE. Mature lacuna structures and metachromatic matrices were also observed in these specimens. GAG contents in experimental group were (5.13 +/- 0.32) mg/g (4 weeks), (5.37 +/- 0.12) mg/g (8 weeks). In contrast, specimens in BMSC group showed mainly fibrous tissue.</p><p><b>CONCLUSION</b>It indicates that it is feasible to create special shaped tissue-engineering cartilage with the permanent internal support using BMSCs as seed cell.</p>

Application of adipose-derived cells in reconstruction of tissue engineered cartilage in vitro / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the feasibility of application of adipose-derived cells (ADCs) in reconstruction of tissue engineered cartilage in vitro.</p><p><b>METHODS</b>Adipose tissue were obtained from human liposuction aspirate (19 cases, 31.5 +/- 5.8 years old). ADCs were isolated by collagenase digestion from liposuction aspirates. 3rd passage cells were seeded into PLGA scaffolds. The copolymer constructs were cultured in conditioned or non-conditioned medium in vitro for 4 weeks. The constructs were evaluated though gross morphology, histology, and immunohistochemistry.</p><p><b>RESULTS</b>The cell-polymer constructs kept its original shape in the induced group, but lost its original shape in the non-induced group. The scaffold group were collapsed. Histologically, the induced groups showed dense cellularity and lacunae-containing cells embedded in a basophilic matrix, while non-induced groups showed connective tissue-like morphology. Collagen and proteoglycan deposition was revealed by Massons's trichome and Safranin' O staining, and minor collagen II expression in the matrix was detected by immunohistochemistry staining in the induced group. They were all negative in the non-induced groups.</p><p><b>CONCLUSIONS</b>Although ADCs included many kinds of cells, it is feasible to use ADCs as seeds cells for reconstruction of tissue engineered cartilage.</p>

Creation of tissue engineered cartilage with internal support / 中华整形外科杂志

<p><b>OBJECTIVE</b>To test the hypothesis that tissue-engineered cartilage can be bioincorporated with a nonreactive, permanent endoskeletal scaffold.</p><p><b>METHODS</b>Chondrocytes obtained from swine articular were seeded onto polyglycolic acids(PGA) scaffold which was incorporated with high-density polyethylene (Medpor). After cultured in vitro for two weeks,the cell-scaffold construct was implanted into subcutaneous pockets on the back of nude mice. Six weeks later,the newly formed cartilage prosthesis was harvested, and a small part of sample was evaluated by gross view, histology, type II collagen immunohistochemistry and biochemistry. PGA scaffold seeded with cells as the control group.</p><p><b>RESULTS</b>The newly formed cartilage was very similar to normal cartilage in both gross view and histology, and jointed Medpor tightly. The center of control group was hollow.</p><p><b>CONCLUSION</b>This pilot technique combining tissue engineering with a permanent success in creating cartilage without "hollow" phenomenon. biocompatible endoskeleton demonstrated</p>

Microarray analysis of dedifferentiation related gene expression of human chondrocytes cultured in vitro / 中华整形外科杂志

<p><b>OBJECTIVE</b>Dedifferentiation of chondrocytes during in vitro expansion is the major cause that limits the potential of chondrocytes for cartilage engineering. This study dissected dedifferentiation mechanism of in vitro cultured human chondrocytes by microarray analysis of gene expression changes.</p><p><b>METHODS</b>Spare human costal cartilage from ear reconstruction patients (n=3, aged 10-20) were digested with collagenase II to isolate human chondrocytes(HCC) and were expanded from P1 to P4. For microarray, RNA was isolated from the P1 and P4 cells and labeled with cy3 and cy5 fluorescence respectively as probes to hybridize a cDNA microarray( about 8300 genes). Those genes with 2-fold difference of expression were selected.</p><p><b>RESULTS</b>Microarray results showed that 56 genes were up-regulated at P4, including proteases, inflammatory factors, growth suppressor and apoptosis genes. Down-regulated 84 genes at P4 include extracellular matrices, protein anabolism, growth factors, cell cycle and cytoskeleton related genes.</p><p><b>CONCLUSIONS</b>These results provide insight into the mechanism of chondrocytes dedifferentiation. The characteristic appearance of dedifferentiation related genes expression includes increased level of stress response and inflammation. The down-regulation of anabolic metabolism related genes and up-regulation of proteases genes leaded to extra cellular matrix decrease. Decreased production of growth factors and increased apoptosis cause decreased cell proliferation of dedifferentiation chondrocytes.</p>

An experimental study on influence of concentration of fetal bovine serum on chondrogenesis of BMSCs / 中华整形外科杂志

<p><b>OBJECTIVE</b>To explore the chondrogenetic effect of induce media containing different concentrations of fetal bovine serum (FBS) on BMSCs differentiation in vitro and provide technical parameters for cartilage engineering in vitro.</p><p><b>METHODS</b>Passage 2 BMSCs of swine were seeded at the density of 5 x 10(7) cells/cm3 to disc-shaped PGA scaffolds with a diameter of 5mm and a thickness of 2mm. After 7days, the scaffolds were induced in media with TGF-beta1, IGF-I, dexamethasone, and different concentrations of FBS: 0% in A group, 5% in B group, and 10% in C group. Specimens were collected after 8 weeks for gross observation, size evaluation, wet weight, glycosaminoglycan (GAG) content, histology assessment, and immunohistology of type II collagen.</p><p><b>RESULTS</b>The compound of C group showed china-white color, hard and fine texture, no obvious change in size and shape, typical lacuna structures, cartilage specific ECM, and significantly higher wet weight and GAG content. The compound of B group showed reduced size, fewer lacuna structures and some cartilage specific ECM. And the compound of A group showed greatly reduced size, soft and loose texture, and no typical lacuna structure or cartilage specific ECM.</p><p><b>CONCLUSIONS</b>FBS was indispensable to chondrogenetic media for in-vitro tissue engineering of cartilage with BMSCs.</p>

Effect of adeno-BMP7 transfection on osteogenesis of BMSCs / 中华整形外科杂志

<p><b>OBJECTIVE</b>To study the effect of adeno-BMP7 transfection on the biology of bone marrow stromal cells (BMSCs).</p><p><b>METHODS</b>Bone marrow was obtained from the goat. The BMSCs were isolated and cultured at the second passage. Once the cells attached and formed a monolayer with 70%-80% confluency, adeno-BMP7 (M.O.I. = 100) was added to the cells. After three days, calcium node was examined with staining; cell-coral compound was replanted subcutaneously.</p><p><b>RESULTS</b>With adeno-BMP7 transfection, BMP7 expression was detected with Western-blot; big calcium nodes were observed with staining. New bone formation was enhanced, which was evaluated by X-ray and histological examinations.</p><p><b>CONCLUSIONS</b>BMSCs transfected with adeno-BMP7 show much stronger osteogenic ability.</p>

Research advances in biochemical structure,biomechanical property and their relationship of articular cartilage / 上海第二医科大学学报

Articular cartilage defects are commonly found in clinics. It is always a great challenge for the repair of cartilage defects due to the limited self-regeneration after injury. Tissue engineering,a newly emerging biotechnique to regenerate cartilage with chondrogenic potential cells and biodegradable scaffolds in vivo and in vitro,provides a promising method to solve the challenge in cartilage defects repair. To regenerate cartilage with favourable structure and function,it is essential to gain a deep insight into the biochemical structure,biomechanical property and their relationship of articular cartilage. This article gives an introduction to the biochemical structure,biomechanical property and their relationship of both native and tissue-engineered cartilage.

Research advances on inducing bone marrow stromal cells chondrogenic differentiation in vitro / 上海第二医科大学学报

The source of seed cells has always been the major problem in cartilage tissue engineering.Bone marrow stromal cells (BMSCs) have gradually become an optimal source of seed cells for cartilage engineering due to their high proliferative potential,multi-lineage differentiation potential and easiness to be obtained with minute trauma.The great challenge is how to get abundant BMSCs with a high purity and how to induce them in vitro into chondrogenic phenotype.This review aims to discuss the various strategies that can induce BMSCs chondrogenic differentiation in vitro so as to offer beneficial reference for constructing cartilage with BMSCs as seed cells.

Construction of tissue engineered porcine corneal stroma with skin fibroblasts / 上海第二医科大学学报

Objective To explore the feasibility of constructing tissue engineered porcine corneal stroma with skin fibroblasts in vivo.Methods Skin fibroblasts were isolated from embryonic porcine,cultured and expanded in vitro.Cells were labeled with green fluorescence protein(GFP) gene by retro-viral infection.Cells at passage 3 were seeded on polyglycolic acid(PGA) non-woven fibers to form a cell-scaffold complex.The complexes were then implanted into porcines' corneal stroma after culturing in vitro for 1 week.Engineered stroma was observed continuously and harvested after 8 weeks for gross and histological evaluation.PGA with corneal stromal cells was served as control. Results The engineered tissue in the stroma gradually became transparent over a period of 8 weeks,showing no difference with the control group.Histologically,the engineered stromal lamellar was relatively regular and similar to the control.The implanted cells were confirmed by GFP expression under fluorescent microscope.By transmission electron microscopy examination, no significant difference in the diameter of collagen fiber was observed between the engineered stroma and normal stroma. Conclusion Tissue engineered corneal stroma may be formed with skin fibroblasts in porcine corneal microenvironment.

Constructing Tissue Engineered Cartilage in Vitro Using a Perfusion-Hydrodynamic Pressure Bioreactor / 上海第二医科大学学报

Objective To explore the feasibility and efficacy of constructing tissue engineered cartilage in vitro(using) a perfusion-hydrodynamic pressure bioreactor. Methods Chondrocytes isolated from swine's auricular cartilage were seeded onto polyglycolic acid(PGA) to be cultured in a three dimensional environment for 1 week.Then the chondrocyte-polymer constructs were divided into two groups: the experimental group and control group(8 constructs in each group).The experimental group was put into the perfusion-hydrodynamic pressure bioreactor to be cultured for another 3 weeks.The parameters of bioreactor were set as follows: flow rate of 100 mL/min,clockwise and anticlockwise 30 min respectively,on/off 8 h/16h,hydrodynamic pressure of 100 kpa with 0.5 Hz for 4 h/d.The control group was cultured with the routine method.Specimens were harvested and analyzed by gross observation,histology,typeⅡcollagen immunohistochemistry and biochemistry after 4 weeks. Results After 4 weeks,gross observation showed cartilage-like tissue was formed in both groups,and tissue wet weight of experimental group and control group were(191.03?18.55) mg and(130.78?10.33) mg,respectively(P

Experimental study of porous TCP to generate tissue-engineered long bone / 生物工程学报

To study bone-forming of a new kind of porous beta-TCP as the scaffold for tissue-engineering, defects at the mid-portion of the left and right ulna were created in dog, the defects were repaired with beta-TCP cylinder coated with BMSCs, and beta-TCP cylinders alone as control. X-rays showed the defects were better bridged by the replant with obscure edge and new bone formed in the canal and at the interface in experimental group after three month of operation, whereas in control group, the replants were obviously deformed into dissociated granule with unequal density with only little new bone formed at the interface. After six month, the defects were bridged by new bone with osteodermatous cavum medullare ossium, but in control group, the defects were bridged by high density in radiography without osteodermatous cavum medullare ossium, the diameter of the ular was obviously less than experimental group. There were significant differences between both groups at month one and two in the development pattern through radionuclide observation. By gross, the diameter of ular was smaller in control group than in experimental at month three, and the replants in control group was difficult to detach from the fibroid tissue around it, but in experimental group, there was much more new bone formation, and the surface was rough for the compound of new bone and beta-TCP undegraded completely. The new bone in experiment had been obviously remodeled at month six, but at this moment, the new bone was of infirmity in volume and form. HE staining of three months demonstrated new bone adhered to the surface on the core of beta-TCP in experimental group, but in control group, at the same place, osteoid was observed with much megacayocytes and capillaries. At month six, beta-TCP disappeared completely with new bone formed in both groups, but the volume and structure of the bone was better in experimental group than in control group. From this study it is concluded that the porous beta-TCP can be combined with BMSCs, and the combination could generate new bone to repair long bone defect.